Methods for Treatment of Stroke or Cerebrovascular Accidents Using an ETB Receptor Agonist

ABSTRACT

Methods of using an ET B  receptor agonist, such as IRL-1620, for the treatment of stroke or cerebrovascular accidents are disclosed. The ET B  receptor agonist is used alone or in combination with a second agent useful in the treatment of stroke or other cerebrovascular accident.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 60/965,591, filed Aug. 21, 2007, incorporated herein byreference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None.

FIELD OF THE INVENTION

The present invention relates to methods of using an endothelin_(B)(Er_(B)) receptor agonist, such as IRL-1620, for the treatment of strokeand cerebrovascular accidents caused, for example, by thrombosis,embolism, or hemorrhage. The ET_(B) receptor agonist can be used aloneor in combination with a thrombolytic agent, like tissue plasminogenactivator, or an ET_(A) antagonist, or an erythropoiesis-stimulatingagent, like erythropoietin, darbepoetin, and epoetin alfa, or an oxygencarrier, like a hemoglobin-based blood substitute and aperfluorocarbon-based blood substitute. Other neuroprotective agentsthat can be used in combination with the ET_(B) receptor agonist includeargatroban, alfimeprase, tenecteplase, ancrod, sildenafil, insulin andits growth factor, magnesium sulfate, human serum albumin, caffeinol(combination of caffeine and alcohol), microplasmin, statins,eptifibatide, tinzaparin, enecadin, citicoline, edaravone, cilostazol,or hypothermia. Using an ET_(B) receptor agonist, alone or incombination with an existing neuroprotective agent, provides bloodsupply, oxygenation, and reduces damage to brain cells.

BACKGROUND OF THE INVENTION

The discovery of endothelin-1 (ET-1), a 21-amino acid peptide, hashelped improve knowledge of local regulation of vascular tone by bloodvessels (M. Yanagisawa et al. (1988) Nature 332(6163):411-5). ET-1 isgenerated in endothelial cells and vascular smooth muscle cells viaconversion of proET-1 to ET-1 in the presence of endothelin convertingenzyme-1 (ECE-1). This conversion from proET-1 to ET-1 is essential foroptimal vasoconstrictor activity of ET-1 (M. Yanagisawa et al. (1988)Nature 332(6163):411-5 and G. D. Johnson et al. (1999) J Biol Chem274(7):4053-8).

ET-1 is released from cultured endothelial cells at a slow basal rate.Due to a high vasoconstrictor potency and long lasting action, thecontinuous release of small amounts of ET-1 from endothelial cellstowards the underlying smooth muscle cells may contribute to themaintenance of vascular tone and blood pressure (T. Miyauchi et al.(1999) Annu Rev Physiol 61:391-415). Under physiological conditions, thebasal tone maintained by ET-1 is balanced by the release of endotheliumderived relaxing factor (EDRF or nitric oxide and prostacyclin) andvasoconstrictor substances (thromboxane) (E. L. Schiffrin (1994) ClinInvest Med 17(6):602-20 and P. B. Persson (1996) Physiol Rev76(1):193-244).

ET and its axis (ET-1, ET-2, ET-3, ET_(A) and ET_(B) receptors) havetriggered considerable efforts to develop ET receptor antagonists havingtherapeutic potential in treating diseases like hypertension, heartfailure, renal diseases, and cancer (A. Gulati et al. (1992) DrugDevelop Res 26:361-387; A. Gulati et al. (1997) Neuropeptides 31(4)301-9; G. Remuzzi et al. (2002) Nat Rev Drug Discov 1(12):986-1001; J.Nelson et al. (2003) Nat Rev Cancer 3(2):110-6; and A. Gulati et al.(2004) J Cardiovasc Pharmacol 44:S483-S486). Several ET_(A) receptorantagonists, e.g., atrasentan, avosentan, clazosentan, darusentan,sitaxsentan, and ZD4054, are in mid to late stage clinical trial.Bosentan, a non-specific ET_(A) and ET_(B) receptor antagonist, has beenmarketed for a few years, and ambrisentan (ET_(A) receptor antagonist)recently was approved for sale by the U.S. Food and Drug Administration(FDA) for a once-daily treatment of pulmonary arterial hypertension.

Intense efforts are devoted to develop ET_(A) receptor antagonists.However, virtually no effort has been expended to develop ET agonists astherapeutic agents. The first proposed therapeutic use of an ET_(B)receptor agonist resulted from a discovery that IRL-1620, a potentET_(B) receptor agonist, selectively enhanced breast tumor perfusion inrats (A. Rai et al. (2003) Cancer Chemother Pharmacol 51(1):21-8; A.Gulati (2003) U.S. Patent Publication 2004/0138121; and A. Gulati (2006)U.S. Patent Publication 2006/0211617). Administration of BQ788, a highlyselective ET_(B) receptor antagonist, blocked the tumor perfusioninduced by IRL-1620 and confirmed the involvement of ET_(B) receptors intumor vasodilation (A. Rai et al. (2005) J Pharm Pharmacol 57(7):869-76and N. V. Rajeshkumar et al. (2005) Breast Cancer Research and Treatment94(3):237-247). The selective enhancement of tumor blood flow resultedin a greater percentage of infused paclitaxel reaching the tumor ascompared to the normal tissues.

In a study conducted in breast tumor rats, IRL-1620 administration priorto paclitaxel resulted in a significant reduction of tumor volume, aswell as a 20% complete remission of tumors, compared to paclitaxeltreated rats (A. Rai et al. (2005) J Pharm Pharmacol 57(7):869-76. andN. V. Rajeshkumar et al. (2005) Breast Cancer Research and Treatment94(3):237-247). See United States Patent Publication Nos. 2004/0138121,2006/0211617, 2006/0257362, and 2007/0032422.

The present invention is directed to a new use for ET_(B) receptoragonists, including IRL-1620, in the treatment of stroke and othercerebrovascular accidents. In particular, it now has been found that anET_(B) receptor agonist significantly increases cerebral bloodperfusion, which is a novel and unexpected finding.

ETs are widely distributed throughout the body and are involved in avariety of physiological functions (A. Gulati et al. (1992) Drug DevelopRes 26:361-387 and J. Nelson et al. (2003) Nat Rev Cancer 3(2):110-6).ETs exert their effects by binding to two distinct types of cell surfacereceptors, ET_(A) and ET_(B) ET_(A) receptors have equal affinity forET-1 and ET-2, and low affinity for ET-3. ET_(B) receptors have equalaffinity for ET-1, ET-2, and ET-3. Pharmacological evidence suggeststhat ET_(B) receptors can be divided into two subtypes, i.e., ET_(B1)receptors present on endothelial cells and ET_(B2) receptors present onsmooth muscle cells (D. P. Brooks et al. (1995) J Cardiovasc Pharmacol26 Suppl 3:S322-5 and A. Leite-Moreira et al. (2004) Am J Physiol HeartCirc Physiol 287(3):H1194-9). Both ET_(A) and ET_(B) receptors belong tothe G protein-coupled receptor (GPCR) family (J. Nelson et al. (2003)Nat Rev Cancer 3(2):110-6). ET_(A) and ET_(B) receptors located onvascular smooth muscle cells, produce vasoconstriction, whereas ET_(B)receptors present on endothelial cells are mainly vasodilatory (G.Remuzzi et al. (2002) Nat Rev Drug Discov 1(12):986-1001).

IRL-1620 (N-Succinyl-[Glu⁹, Ala^(11,15)] Endothelin 1) is a syntheticanalogue of ET-1, i.e., a fragment of ET-1 having amino acids 8-21 ofET-1. IRL-1620 is a highly selective endothelin B receptor agonist,being 120,000 times more selective to ET_(B) receptors than to ET_(A)receptors (M. Takai et al. (1992) Biochem Biophys Res Commun184(2):953-9). IRL-1620 has a molecular formula of C₈₆H₁₁₇N₁₇O₂₇ and amolecular weight of 1820.95. The molecular structure of IRL-1620, asillustrated in FIG. 1, is an amino acid sequence ofSuc-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp (SEQ IDNO:1).

Pharmacological Effects of IRL-1620

IRL-1620, like endothelins, can produce both vasodilation andvasoconstriction. Interaction of IRL-1620 with ET_(B) receptors onendothelial cells leads to vasodilation, whereas an interaction withET_(B) receptors on smooth muscle cells leads to vasoconstriction.Furthermore, primary activation of ET_(B) receptors by IRL-1620 can leadto autocrine/paracrine ET-1 release that subsequently activates bothET_(A) and ET_(B) receptors (S. Noguchi, et al. (1996) Br J Pharmacol118(6):1397-402). Thus, the net effect of IRL-1620 is related to anumber of factors, including the type of tissue, the species, and thephysiological conditions. There have been a number of studies onpharmacological effects of IRL-1620 because it is a highly selectiveagonist of ET_(B) receptors and often is used to delineate the role ofET_(B) receptors in a given physiological situation. Some of thesestudies summarized below show that the vasoconstrictive effects ofIRL-1620 are much less pronounced than those of ET-1. Other ET_(B)receptor agonists known to persons skilled in the art producepharmacological effects similar to those of IRL-1620, with the neteffect also being related to the ability of a specific compound toselectively agonize ET_(B) receptors.

Systemic Hemodynamic Effects

IRL-1620 exhibits systemic hemodynamic effects, including transientvasodilation and sustained vasoconstriction, in anesthetized rats (B.Palacios et al. (1997) Br J Pharmacol 122(6):993-8 and S. W. Leung etal. (2002) J Cardiovasc Pharmacol 39(4):533-43), in an open-chest ratmodel (M. E. Beyer et al. (1995) J Cardiovasc Pharmacol 26 Suppl3:S150-2), and in normal and cardiomyopathic hamsters (J. C. Honore etal. (2002) Clin Sci (Lond) 103 Suppl 48:280S-283S). The vasoconstrictiveeffects of IRL-1620 are less pronounced compared to those of ET-1(Palacios et al. (1997) Br J Pharmacol 122(6):993-8; J. C. Honore et al.(2002) Clin Sci (Lond) 103 Suppl 48:280S-283S; and S. W. Leung et al.(2002) J Cardiovasc Pharmacol 39(4):533-43) and IRL-1620 had a positiveinotropic effect (M. E. Beyer et al. (1995) J Cardiovasc Pharmacol 26Suppl 3:S150-2).

Regional Hemodynamic Effects

IRL-1620 causes renal vasodilation in anesthetized dogs upon intrarenalarterial perfusion (T. Yukimura et al. (1994) Eur J Pharmacol264(3):399-405) and pulmonary vasodilation in neonatal lambs uponintrapulmonary arterial injection (J. Wong et al. (1995) J CardiovascPharmacol 25(2):207-15). A pulmonary vasodilatory effect of IRL-1620also is observed in isolated perfused rat lungs (M. Muramatsu et al.(1999) Am J Physiol 276(2 Pt 1):L358-64). Injection of IRL-1620 into thecircumflex coronary artery of anesthetized goats does not cause coronaryvasoconstriction, whereas ET-1 administered similarly caused coronaryvasoconstriction (J. L. Garcia et al. (1996) Eur J Pharmacol315(2):179-86).

Effect on Respiratory Airway Smooth Muscles

Intravenous administration of IRL-1620 to anesthetized,artificially-ventilated guinea pigs resulted in bronchoconstriction in abiphasic manner (S. Noguchi et al. (1996) Br J Pharmacol118(6):1397-402). The second phase of bronchoconstriction probably isdue to the activation of ET_(B) receptors by IRL-1620 leading toautocrine/paracrine release of ET-1 that subsequently activated bothET_(A) and ET_(B) receptors (S. Noguchi et al. (1996) Br J Pharmacol118(6):1397-402).

Experimental Studies on Human Tissues

In vitro, IRL-1620 causes contraction of human internal mammary arterialsegments, but not human radial arterial segments (J. J. Liu, et al.(1996) Clin Sci (Loud) 90(2):91-6). The contractile effect of IRL-1620on internal mammary arteries reached a maximum of 20% of that obtainedwith ET-I or noradrenaline. Further increases in concentration ofIRL-1620 caused relaxation of the contracted arteries. IRL-1620 also hada contractile effect on human bronchial rings in a biphasic manner (T.Takahashi et al. (1997) Eur J Pharmacol 324(2-3):219-22).

Clinical Studies

To date, IRL-1620 has not been administered to humans. However, a phaseI, open label, ascending dose study of the safety, tolerability,pharmacokinetics, and pharmacodynamics of IRL-1620 in patients withrecurrent or progressive carcinoma (NCT00613691) is ongoing.Furthermore, a number of human studies have been conducted with ET-1, amuch more potent vasoconstrictive agent than IRL-1620, as demonstratedin animal studies (B. Palacios et al. (1997) Br J Pharmacol 122(6):993-8and S. W. Leung et al. (2002) J Cardiovasc Pharmacol 39(4):533-43).Administration of ET-1 to human subjects by perfusion at doses rangingfrom 1 to 20 ng/kg/min caused dose-dependent systemic vasoconstrictionand consequential changes in hemodynamic parameters (D. Kiely et al.(1997) Cardiovasc Res 33(2):378-86; A. Franco-Cereceda et al. (1999)Scand Cardiovasc J 33(3):151-6; and F. Kiefer et al. (2000) Exp ClinEndocrinol Diabetes 108(5):378-81), but did not produce any seriousadverse events.

Intravenous administration of ET-1 also causes coronary vasoconstriction(J. Pernow et al. (1996) Circulation 94(9):2077-82). However, coronaryvasoconstriction may not be expected with IRL-1620 in humans. It hasbeen shown that, in human coronary arteries, ET_(B) receptors are absentor present at very low levels, and therefore, would make minimalcontribution toward coronary vasoconstriction (W. A. Bax et al. (1994)Br J Pharmacol 113(4):1471-9; A. P. Davenport et al. (1995) J CardiovascPharmacol 26 Suppl 3:S265-7; A. P. Davenport et al. (1994) Br JPharmacol 111(1):4-6; W. A. Bax et al. (1993) Naunyn Schmiedebergs ArchPharmacol 348(4):403-10; A. P. Davenport et al. (1995) J CardiovascPharmacol 22 Suppl 8:522-5; and O. Saetrum Opgaard et al. (1996) RegulPept 63(2-3):149-56).

Human studies also were conducted with an endothelin agonist,sarafotoxin S6c, which is less selective for ET_(B) receptors thanIRL-1620. On infusion into brachial artery, sarafotoxin S6c showed lessreduction in forearm blood flow compared to ET-1 (W. G Haynes et al.(1995) Circulation 92(3): 357-63). Thus, any vasoconstrictive effects ofIRL-1620 in humans are expected to be less than those observed with ET-1and other endothelin agonists administered to humans to date.

Effect on Cerebral Blood Vessels

Endothelin has been implicated in a number of cerebrovascular disorders,including subarachnoid hemorrhage (R. Suzuki et al. (1992) J Neurosurg77(1):96-100) and ischemic stroke (I. Ziv et al. (1992) Stroke23(7):1014-6). It has been found that ET_(A) receptor antagonistsrelieve chronic cerebral vasospasm (M. Clozel et al. (1993) Life Sci52(9):825-34; S. Itoh et al. (1993) Biochem Biophys Res Commun195(2):969-75; H. Nirei et al. (1993) Life Sci 52(23):1869-74; and R. N.Willette et al. (1994) Stroke 25(12):2450-5; discussion 2456). Studieshave been performed to characterize endothelin receptors in the cerebralblood vessels. ET_(A) receptors were found to mediate contraction inhuman cerebral, meningeal, and temporal arteries (M. Adner et al. (1994)J Auton Nery Syst 49 Suppl:S117-21) and a marked ET_(B)receptor-mediated relaxation was obtained with ET-3 when ET_(A) receptoractivity was blocked using FR139317 (IUPAC Name:(2R)-2-[[(2R)-2-[[(2S)-2-(azepane-1-carbonylamino)-4-methylpentanoyl]amino]-3-(1-methylindol-3-yl)propanoyl]amino]-3-pyridin-2-ylpropanoicacid) in precontracted human temporal arteries (G. A. Lucas, et al.(1996). Peptides 17(7): 1139-44).

Overall, a need still exists in the art to identify agents, orcombinations of agents, that effectively treat strokes and othercerebrovascular accidents. To date, no report exists on the effect ofIRL-1620 on cerebral circulation, and the present disclosure is thefirst reporting that IRL-1620 increases cerebral blood perfusion, asmeasured with laser-Doppler perfusion method.

SUMMARY OF THE INVENTION

The present invention is directed to administration of an ET_(B)receptor agonist in the treatment of strokes and other cardiovascularaccidents. Accordingly, one embodiment of the present invention is toprovide a method of treating strokes and other cerebrovascular accidentscomprising administering to a mammal in need thereof a therapeuticallyeffective amount of an ET_(B) receptor agonist.

Another embodiment of the present invention is to provide a compositioncomprising an ET_(B) receptor agonist useful in the treatment of strokesand other cerebrovascular accidents. In particular, the presentinvention is directed to compositions containing an ET_(B) receptoragonist, and to methods of administering the composition to treatstrokes and other cerebrovascular accidents. In yet another embodiment,the composition further comprises a pharmaceutically acceptable carrier.

Another embodiment of the present invention is to provide a compositioncomprising (a) an ET_(B) receptor agonist, (b) a second therapeuticagent useful in the treatment of stroke or other cerebrovascularaccident, and (c) an optional excipient and/or pharmaceuticallyacceptable carrier.

In a further embodiment, the invention provides for use of a compositioncomprising an ET_(B) receptor agonist and an optional neuroprotectiveagent for the manufacture of a medicament for treating stroke and othercerebrovascular accidents in an individual in need thereof.

Still another embodiment of the present invention is to provide a kitfor human pharmaceutical use, comprising (a) a container, (b1) apackaged composition comprising an ET_(B) receptor agonist and,optionally, (b2) a packaged composition comprising a neuroprotectiveagent useful in the treatment of stroke or other cerebrovascularaccident, and (c) a package insert containing directions for use of thecomposition or compositions, administered simultaneously orsequentially, in the treatment of stroke and/or other cerebrovascularaccidents.

In one embodiment, the present invention provides a method of treating apatient with stroke or cerebrovascular accident comprising administeringa therapeutically effective amount of an endothelin_(B) (ET_(B))receptor agonist to the patient in need thereof. The stroke orcerebrovascular accident can be caused, for example, by thrombosis,embolism, or hemorrhage. In one preferred embodiment, the ET_(B)receptor agonist comprises N-Succinyl-[Glu⁹, Ala^(11,15)] Endothelin 1(i.e., IRL-1620).

The ET_(B) receptor agonist can be administered alone, or in combinationwith a second therapeutic agent useful in a treatment of stroke or othercerebrovascular accident, such as one or more neuroprotective agent,like a thrombolytic agent (such as, but not limited to, tissueplasminogen activator), or an ET_(A) antagonist, such as, but notlimited to, sulfosoxazole, clazosentan, atrasentan, tezosentan,bosentan, sitaxsentan, enrasentan, BMS 207940, BMS 193884, BMS 182874, J104132, VML 588/Ro 61 1790, T-0115, TAK 044, BQ 788, TBC2576, TBC3214,PD180988, ABT 546, SB247083, RPR118031A, and BQ123), anerythropoiesis-stimulating agent (such as erythropoietin, darbepoetin,and epoetin alfa), or an oxygen carrier (such as a hemoglobin-basedblood substitute or a perfluorocarbon based blood substitute). Otherneuroprotective agents that can be administered in combination with theET_(B) receptor agonist include, but are not limited to, argatroban,alfimeprase, tenecteplase, ancrod, sildenafil, insulin and its growthfactor, magnesium sulfate, human serum albumin, caffeinol (combinationof caffeine and alcohol), microplasmin, statins, eptifibatide,tinzaparin, enecadin, citicoline, edaravone, cilostazol, hypothermia,and mixtures thereof.

The ET_(B) receptor agonist and the second therapeutic agent can beadministered together as a single-unit dose or separately as multi-unitdoses, wherein the ET_(B) receptor agonist is administered before thesecond therapeutic agent or vice versa. It is envisioned that one ormore dose of the ET_(B) receptor agonist or one and/or more dose of thesecond therapeutic agent can be administered.

In an embodiment, the ET_(B) receptor agonist and neuroprotectivetherapeutic agent are administered simultaneously. In relatedembodiments, the ET_(B) receptor agonist and neuroprotective therapeuticagent are administered from a single composition or from separatecompositions. In a further embodiment, the ET_(B) receptor agonist andneuroprotective agent are administered sequentially. The ET_(B) receptoragonist, as used in the present invention, can be administered in anamount of about 0.005 to about 500 micrograms per dose, about 0.05 toabout 250 micrograms per dose, or about 0.5 to about 50 micrograms perdose. Alternatively, the ET_(B) receptor agonist can be administered inan amount of about 0.005 to about 50 micrograms per kilogram per mininfusion, or about 0.05 to about 5 micrograms per kilogram per mininfusion.

These and other aspects and features of the present invention willbecome apparent from the following drawings and detailed description ofthe preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the molecular structure of IRL-1620;

FIG. 2 shows the effect of IRL-1620 (3 nmol/kg, iv) on cerebral bloodperfusion of urethane anesthetized rat using Laser Doppler Flowmetry,wherein IRL-1620 significantly increased cerebral blood perfusioncompared to baseline;

FIG. 3 shows the effect of IRL-1620 (3 nmol/kg, iv) on cerebral andrenal blood perfusion of urethane anesthetized rat using Laser DopplerFlowmetry, wherein IRL-1620 significantly increased cerebral bloodperfusion and decreased renal blood perfusion compared to baseline;

FIG. 4 shows the effect of IRL-1620 (3 nmol/kg, iv or 5 μg/kg, iv) onblood pressure and heart rate of urethane anesthetized rat, i.e., atransient decrease in blood pressure and an increase in heart rate whichreturned to normal quickly;

FIG. 5 shows the effect of IRL-1620 (5 μg/kg, i.v.) and ET-1 (0.75μg/kg, i.v.) on cerebral blood flow, wherein IRL-1620 caused an increasein cerebral blood flow that persisted for about 60 minutes;

FIG. 6 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (ET_(A) antagonists) (1 μg/kg, i.v.) on the effect of IRL-1620(5 μg/kg, i.v.) on cerebral blood flow;

FIG. 7 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of ET-1 (0.75 μg/kg, i.v.) oncerebral blood flow;

FIG. 8 shows the effect of IRL-1620 (5 μg/kg, i.v.) and ET-1 (0.75μg/kg, i.v.) on renal blood flow, wherein IRL-1620 elicited a decreasein renal blood flow that persisted for about 15 minutes;

FIG. 9 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and, BQ788 (1 μg/kg, i.v.) on the effect of IRL-1620 (5 μg/kg, i.v.) onrenal blood flow;

FIG. 10 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of ET-1 (0.75 μg/kg, i.v.) onrenal blood flow, wherein no pretreatment significantly altered theeffect of ET-1 on renal blood flow;

FIG. 11 shows the effect of administration of IRL-1620 (5.0 μg/kg, i.v.)and ET-1 (0.75 μg/kg, i.v.) on mean arterial pressure;

FIG. 12 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of IRL-1620 (5 μg/kg, i.v.) onmean arterial pressure; and

FIG. 13 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of ET-1 (0.75 μg/kg, i.v.) onmean arterial pressure, wherein pretreatment with BQ788 and BMS failedto alter the effect of ET-1 on mean arterial pressure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is described in connection with preferredembodiments, however, it should be appreciated that the invention is notlimited to the disclosed embodiments. It is understood that, given theabove description of the embodiments of the invention, variousmodifications can be made by one skilled in the art. Such modificationsare intended to be encompassed by the claims below.

As used herein, the terms “endothelin_(B) receptor agonist”, “ET_(B)receptor agonist”, and “ET_(B) agonist” are used interchangeably.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. Although not precluded, treating a disorder or condition doesnot require that the disorder, condition or symptoms associatedtherewith be completely eliminated. As used herein, the terms “treat,”“treating,” “treatment,” and the like may include “prophylactictreatment” which refers to reducing the probability of redeveloping adisorder or condition, or of a recurrence of a controlled disease orcondition, in a subject who does not have, but is at risk of or issusceptible to redeveloping a disorder or condition or a recurrence of adisorder or condition.

The term “therapeutically effective amount” or “effective dose” as usedherein refers to an amount of the active ingredient(s) that is(are)sufficient, when administered by a method of the invention, toefficaciously deliver the agents for the treatment of stroke or acerebrovascular accident.

The term “container” means any receptacle and closure therefor suitablefor storing, shipping, dispensing, and/or handling a pharmaceuticalproduct.

The term “insert” means information accompanying a pharmaceuticalproduct that provides a description of how to administer the product,along with the safety and efficacy data required to allow the physician,pharmacist, and patient to make an informed decision regarding use ofthe product. The package insert generally is regarded as the “label” fora pharmaceutical product.

“Concurrent administration,” “administered in combination,”“simultaneous administration” and similar phrases mean that acomposition comprising two or more agents are administered concurrentlyto the subject being treated. By “concurrently,” it is meant that eachagent is administered simultaneously or sequentially in any order atdifferent points in time. However, if not administered simultaneously,they are, in one aspect, administered sufficiently closely in time so asto provide the desired treatment effect of the combination of agents.Suitable dosing intervals and dosing order of the agents will: bereadily apparent to those skilled in the art. It is also contemplatedthat two or more agents are administered in separate compositions, andin one aspect, one agent is administered prior to or subsequent toadministration of the other agent. Prior administration refers toadministration of the agents within the range of one day (24 hours)prior to treatment up to 30 minutes before treatment. It is furthercontemplated that one agent is administered subsequent to administrationof the other agent. Subsequent administration is meant to describeadministration from 30 minutes after administration of the other agentup to one day (24 hours) after administration of the first agent. Within30 minutes to 24 hours may include administration at 30 minutes, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20 or 24 hours.

The use of the terms “a”, “an”, “the”, and similar referents in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The use of any and all examples, orexemplary language (e.g., “such as”) provided herein, is intended merelyto better illustrate the invention and is not a limitation on the scopeof the invention unless otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementas essential to the practice of the invention.

It has been estimated 40 million Americans will be age 65 or older in2010. An aging population undoubtedly will result in an increased numberof cases of chronic diseases, including coronary artery disease, heartfailure, and stroke. In addition, an explosive increase in theprevalence of obesity and type 2 diabetes has occurred, and relatedcomplications, like hypertension, hyperlipidemia, and atheroscleroticvascular disease, also are expected to increase.

Each year, about 700,000 people experience a new or recurrent stroke.About 500,000 are first attacks and about 200,000 are recurrent attacks.On average, someone in the United States has a stroke every 45 seconds.Also on average, every 3 to 4 minutes someone dies of a stroke. Of allstrokes, 87% are ischemic. Intracerebral and subarachnoid hemorrhagestrokes account for the remainder. When considered separately from othercardiovascular diseases, stroke ranks third among all causes of death,behind diseases of the heart and cancer. From 1979 to 2004; the numberof inpatient discharges from short-stay hospitals with stroke as thefirst listed diagnosis increased 21% to 906,000. The estimated directand indirect cost of stroke for 2007 was $62.7 billion.

Each year, about 46,000 more women than men have a stroke. Male strokeincidence rates are greater than female rates at younger ages, but notat older ages. The male/female incidence was 1.25 in those 55 to 64years of age, 1.50 in those 65 to 74 years of age, 1.07 in those 75 to84 years of age, and 0.76 in those above 85 years of age. Among AmericanIndians/Alaska natives age 18 and older, 5.1% have had a stroke. Amongblacks or African Americans the rate was 3.2%, among whites the rate was2.5%, and among Asians the rate was 2.4%.

The U.S. Food and Drug Administration (FDA) has approved theclot-dissolving drug tissue plasminogen activator (tPA) to treat strokescaused by blood clots, which cause about 80 percent of all strokes. tPAdissolves the clot and restores blood flow to the brain. tPA carries arisk of bleeding in the brain, but its benefits outweigh the risks whenprescribed and administered properly.

It now has been discovered that administration of an ET_(B) receptoragonist, like IRL-1620, together with an optional, neuroprotectiveagent, like tissue plasminogen activator, can greatly reduce theneurological deficit in patients suffering from stroke by increasingblood perfusion and reducing damage to the brain.

The methods described herein benefit from the use of an ET_(B) agonistand an optional neuroprotective agent useful in the treatment andmanagement of strokes and other cerebrovascular accidents. The ET_(B)receptor agonist and optional neuroprotective agent can be administeredsimultaneously or sequentially to achieve the desired effect.

The present invention therefore is directed to compositions and methodsof treating strokes and other cerebrovascular accidents. The presentinvention also is directed to pharmaceutical compositions comprising anET_(B) receptor agonist and a second therapeutic agent useful in thetreatment of strokes and other cerebrovascular accidents, e.g., aneuroprotective agent. Further provided are kits comprising an ET_(B)receptor agonist and, optionally, a second therapeutic agent useful inthe treatment of strokes and other cerebrovascular accidents, packagedseparately or together, and an insert having instructions for usingthese active agents.

As demonstrated below, IRL-1620 increases cerebral blood perfusion andcan be used to increase blood perfusion in patients suffering fromstroke. IRL-1620 and other ET_(B) agonists can be particularly useful intreating conditions, like cerebral ischemia, where an increase incerebral blood flow can help in reducing the penumbra and can greatlyreduce the neurological deficits due to stroke.

In accordance with the present invention, it has been found that aselective ET_(B) receptor agonist, as exemplified by IRL-1620, can beused for the treatment of stroke and other cerebrovasuclar accidents. AnET_(B) receptor agonist utilized in the present invention is notlimited, and can be any ET_(B) receptor agonist known in the art.Preferably, the ET_(B) receptor agonist is selective for the ET_(B)receptor, i.e., is more selective to ET_(B) receptors than ET_(A)receptors by a factor of at least 10³.

Specific examples of ET_(B) agonists useful in the present inventioninclude, but are not limited to, IRL-1620, ET-3, sarafotoxin 6c, BQ3020,Ala(1, 3, 11, 15)ET-1, and mixtures thereof. In particular, sarafotoxin6c (i.e., SFT6C) has an amino acid sequence:

(SEQ ID NO: 2) H-Cys-Thr-Cys-Asn-Asp-Met-Thr-Asp-Glu-Glu-Cys-Leu-Asn-Phe-Cys-His-Gln-Asp-Val-Ile-Trp-OH;(Disulfide bridge: 1-15 and 3-11)and a molecular weight of 2515.6.

IRL-1620, also termed N-Succinyl-[Glu⁹, Ala^(11,15)]-Endothelin 1fragment 8-21, has amino acid sequenceSuc-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp (SEQ ID NO:1); a molecular formula of C₈₆H₁₁₇N₁₇O₂₇; and a molecular weight of1820.95.

Endothelin 3 (ET-3) has an amino acid sequence ofCys-Thr-Cys-Phe-Thr-Tyr-Lys-Asp-Lys-Glu-Cys-Val-Tyr-Tyr-Cys-His-Leu-Asp-Ile-Ile-Trp[Disulfide Bridges: 1-15; 3-11] (SEQ ID NO: 3); a molecular formula ofC₁₂₁H₁₆₈N₂₆O₃₃S₄; and a molecular weight of 2643.04.

BQ3020, also termed (N-Ac-Ala(11,15)-endothelin-1 (6-21)) andN-Aceytyl-[Ala11,15]-Endothelin 1 fragment 6-21, has an amino acidsequence ofAc-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp (SEQID NO: 4); a molecular formula of C₉₆H₁₄₀N₂O₂S₅; and a molecular weightof 2008.32.

Ala(1,3,11,15)ET-1 (CAS Number 121204-87-3) has an amino acid sequenceofAla-Ser-Ala-Ser-Ser-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp(SEQ ID NO: 5), a molecular formula of C₁₀₉H₁₆₃N₂₅O₃₂S; and a molecularweight of 2367.67.

Thus, in one embodiment, the present invention discloses a method ofpreventing or treating an individual suffering from a stroke or othercerebrovascular accident comprising administering a therapeuticallyeffective amount of an ET_(B) agonist to the individual. The stroke orcerebrovascular accident can be caused, for example, by thrombosis,embolism, or hemorrhage. In a preferred embodiment, the ET_(B) receptoragonist comprises N-Succinyl-[Glu⁹, Ala^(11,15)] Endothelin 1(IRL-1620).

Pharmaceutical compositions containing the ET_(B) agonist are suitablefor administration to humans. Typically, the pharmaceutical compositionsare sterile, and contain no toxic, carcinogenic, or mutagenic compoundsthat would cause an adverse reaction when administered.

The method of the present invention can be accomplished using an ET_(B)agonist. The ET_(B) agonist can be administered as the neat compound, oras a pharmaceutical composition. Administration of the pharmaceuticalcomposition, or neat ET_(B) agonist, can be performed during or afterthe onset of stroke or other cerebrovascular accident.

The ET_(B) agonists also can be administered in conjunction with one ormore second therapeutic agent useful in the treatment of stroke or othercerebrovascular accident. The second therapeutic agent is different froman ET_(B) agonist. The ET_(B) agonist and second therapeutic agent canbe administered simultaneously or sequentially. In addition, the ET_(B)agonist and second therapeutic agent can be administered from a singlecomposition or two separate compositions. Preferred second therapeuticagents comprise a neuroprotective agent.

Nonlimiting examples of second therapeutic agents include,neuroprotective agents like, but are not limited to, a thrombolyticagent (such as, but not limited to, tissue plasminogen activator), anET_(A) antagonist (such as, but not limited to, sulfosoxazole,clazosentan, atrasentan, tezosentan, bosentan, sitaxsentan, enrasentan,BMS 207940, BMS 193884, BMS 182874, J 104132, VML 588/Ro 61 1790,T-0115, TAK 044, BQ 788, TBC2576, TBC3214, PD180988, ABT 546, SB247083,RPR118031A, and BQ123), an erythropoiesis-stimulating agent (such aserythropoietin, darbepoetin, and epoetin alfa), or an oxygen carrier(such as, a hemoglobin-based blood substitute or a perfluorocarbon basedblood substitute). Other neuroprotective agents that can be administeredin combination with the ET_(B) receptor agonist include, but are notlimited to, argatroban, alfimeprase, tenecteplase, ancrod, sildenafil,insulin and its growth factor, magnesium sulfate, human serum albumin,caffeinol (combination of caffeine and alcohol), microplasmin, statins,eptifibatide, tinzaparin, enecadin, citicoline, edaravone, cilostazol,hypothermia, or mixtures thereof.

The neuroprotective agent is administered in an amount to provide itsdesired therapeutic effect. The effective dosage range for eachneuroprotective agent is known in the art, and the neuroprotective agentis administered to an individual in need thereof within such establishedranges.

The ET_(B) receptor agonist and the neuroprotective agent can beadministered together as a single-unit dose or separately as multi-unitdoses, wherein the ET_(B) receptor agonist is administered before theneuroprotective agent or vice versa. One or more dose of the ET_(B)receptor agonist and/or one or more dose of the neuroprotective agentcan be administered. It is further contemplated that administration ofthe agents occurs within 30 minutes up to about one day (24 hours).

An ET_(B) receptor agonist used in a method present invention can beadministered in an amount of about 0.005 to about 500 micrograms perdose, about 0.05 to about 250 micrograms per dose, or about 0.5 to about50 micrograms per dose. For example, the ET_(B) agonist can beadministered, per dose, in an amount of about 0.005, 0.05, 0.5, 5, 50,or 500 micrograms, including all doses between 0.005 and 500 micrograms.

Alternatively, the ET_(B) receptor agonist can be administered in anamount of about 0.005 to about 50 micrograms per kilogram per mininfusion, or from about 0.05 to about 5 micrograms per kilogram per mininfusion. For example, the ET_(B) agonist can be administered in anamount of about 0.005, 0.05, 0.5, 5, or 50 in micrograms per kilogramper min infusion.

The ET_(B) agonist can be formulated in suitable excipients for oraladministration or for parenteral administration. Such excipients arewell known in the art. The ET_(B) agonists typically are present in sucha composition in an amount of about 0.1% to about 75% by weight of thecomposition.

The ET_(B) agonists can be administered by any suitable route, forexample by oral, buccal, inhalation, sublingual, rectal, vaginal,intracisternal or intrathecal through lumbar puncture, transurethral,nasal, percutaneous, i.e., transdermal, or parenteral (includingintravenous, intramuscular, subcutaneous, intracoronary, intradermal,intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar,intrapulmonary injection and/or surgical implantation at a particularsite) administration. Parenteral administration can be accomplishedusing a needle and syringe or using a high pressure technique.

The pharmaceutical compositions include those wherein the ET_(B) agonistis administered in an effective amount to achieve its intended purpose.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

The exact formulation, route of administration, and dosage is determinedby an individual physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide levels ofthe ET_(B) agonist that is sufficient to maintain therapeutic orprophylactic effects. The amount of pharmaceutical compositionadministered is dependent on the subject being treated, on the subject'sweight, the severity of the affliction, the manner of administration,and the judgment of the prescribing physician.

Specifically, for administration to a human in the treatment of strokeor other cerebrovascular accident, oral dosages of an ET_(B) agonist,individually generally are about 0.005 to about 500 micrograms daily foran average adult patient (70 kg), typically one dose per day or dividedinto two to three doses per day. Thus, for a typical adult patient,individual doses contain about 0.005 to about 500 micrograms ET_(B)agonist, in a suitable pharmaceutically acceptable vehicle or carrier,for administration in single or multiple doses, once or several timesper day. Dosages for intravenous, buccal, or sublingual administrationtypically are about 0.005 to about 250 micrograms/kg per single dose asrequired. In practice, the physician determines the actual dosingregimen that is most suitable for an individual patient, and the dosagevaries with the age, weight, and response of the particular patient. Theabove dosages are exemplary of the average case, but there can beindividual instances in which higher or lower dosages are merited, andsuch are within the scope of this invention.

The ET_(B) agonists can be administered alone, or in admixture with apharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. Pharmaceuticalcompositions for use in accordance with the present invention thus canbe formulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries thatfacilitate processing of the ET_(B) agonists into preparations that canbe used pharmaceutically.

These pharmaceutical compositions can be manufactured in a conventionalmanner, e.g., by conventional mixing, dissolving, granulating,dragee-making, emulsifying, encapsulating, entrapping, or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount of theET_(B) agonists are administered orally, the composition typically is inthe form of a tablet, capsule, powder, solution, or elixir. Whenadministered in tablet form, the composition additionally can contain asolid carrier, such as a gelatin or an adjuvant. The tablet, capsule,and powder contain about 1% to about 95% of an ET_(B) agonist, andpreferably from about 1% to about 50% ET_(B) agonist. When administeredin liquid form, a liquid carrier, such as water, Petroleum, or oils ofanimal or plant origin, can be added. The liquid form of the compositioncan further contain physiological saline solution, dextrose or othersaccharide solutions, or glycols. When administered in liquid form, thecomposition contains about 0.1% to about 90% by weight of ET_(B)agonists, and preferably about 1% to about 50% of ET_(B) agonists.

When a therapeutically effective amount of the ET_(B) agonist isadministered by intravenous, cutaneous, or subcutaneous injection, thecomposition is in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such parenterally acceptablesolutions, having due regard to pH, isotonicity, stability, and thelike, is within the skill in the art. A preferred composition forintravenous, cutaneous, or subcutaneous injection typically contains, anisotonic vehicle. Preferably, the ET_(B) agonist or compositioncontaining the ET_(B) agonist is administered by intravenous or bolusinjection, or infusion with other fluids over a 10-30 minute span orover several hours.

Suitable ET_(B) agonists can be readily combined with pharmaceuticallyacceptable carriers well-known in the art. Such carriers enable theactive agents to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by adding the ET_(B) agonist to a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients include, forexample, fillers and cellulose preparations. If desired, disintegratingagents can be added.

The ET_(B) agonists can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active agent in water-soluble form.Additionally, suspensions of the ET_(B) agonists can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils or synthetic fatty acid esters. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions. Alternatively, a present composition can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

The ET_(B) agonists also can be formulated in rectal compositions, suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases. In addition to the formulations described previously,the ET_(B) agonists also can be formulated as a depot preparation. Suchlong-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the ET_(B) agonists can be formulated withsuitable polymeric or hydrophobic materials (for example, as an emulsionin an acceptable oil) or ion exchange resins.

In particular, the ET_(B) agonists can be administered orally, buccally,or sublingually in the form of tablets containing excipients, such asstarch or lactose, or in capsules or ovules, either alone or inadmixture with excipients, or in the form of elixirs or suspensionscontaining flavoring or coloring agents. Such liquid preparations can beprepared with pharmaceutically acceptable additives, such as suspendingagents. The ET_(B) agonists also can be injected parenterally, forexample, intravenously, intramuscularly, subcutaneously, orintracoronarily. For parenteral administration, the endothelin agonistsare best used in the form of a sterile aqueous solution which cancontain other substances, for example, salts or monosaccharides, such asmannitol or glucose, to make the solution isotonic with blood.

As an additional embodiment, the invention includes kits which compriseone or more compounds or compositions packaged in a manner thatfacilitates their use to practice methods of the invention. In oneembodiment, the kit includes a compound or composition described hereinas useful for practice of a method (e.g., a composition comprising anET_(B) agonist and an optional neuroprotective agent), packaged in acontainer, such as a sealed bottle or vessel, with a label affixed tothe container or included in the kit that describes use of the compoundor composition to practice the method of the invention Preferably, thecompound or composition is packaged in a unit dosage form. The kitfurther can include a device suitable for administering the compositionaccording to the intended route of administration.

It has been found that an ET_(B) receptor agonist, like IRL-1620, cantreat stroke and other cerebrovascular accidents. Tests and data hereinshow that ET_(B) agonists are useful agents to treat stroke and othercerebrovascular accidents.

Experimental Procedures and Results

IRL-1620, a highly selective ET_(B) receptor agonist, transientlyincreases tumor blood flow and has been shown to enhance tumor deliveryand efficacy of anticancer drugs. A phase 1 clinical trial has recentlybegun for the use of IRL-1620 in patients with recurrent or progressivecarcinoma.

Although known to increase tumor blood flow due to the unique structureof tumor vasculature, the effect of administration of IRL-1620 on otherareas of the body that display distinctive vascular structures,specifically the cerebral blood vessels, has not been studied. Thepresent studies were conducted to determine the alteration in cerebralblood flow caused by intravenous administration of the ET_(B) receptoragonist 1RL-1620.

Methods

Male Sprague-Dawley rats weighing 323±4 grams (g) were anesthetized withurethane (ethyl carbamate, Sigma Aldrich, St. Louis, Mo.) dissolved inisotonic saline (0.9% NaCl, Hospira, Inc., Lake Forest, Ill.). The ratswere administered a dose of 0.15 g per 100 g body weight viaintraperitoneal (i.p.) injection.

Blood Pressure and Heart Rate: The femoral vein and artery werecannulated. The arterial cannula was connected to a Gould P23 IDpressure transducer for recording the blood pressure on a Grass P7Dpolygraph through a 7PI preamplifier. The heart rate was recordedthrough a 7P4B Grass tachograph, triggered from blood pressure signals.Drugs were administered via the venous cannula.

Cerebral Perfusion: A burr hole was drilled into the rat skull about 2millimeters (mm) to the left of midline. Cerebrovascular perfusion wasmeasured via a fiber optic probe (PF407) applied to the surface of therat brain. The probe was connected to a Periflux PF2b 4000 Laser DopplerFlowmetry unit (Perimed, Stockholm, Sweden).

Renal Perfusion: The right kidney was dissected retroperitoneally. Renalperfusion was measured via a fiber optic probe (PF408) applied to thesurface of the rat kidney. The probe was connected to a Periflux PF2b4000 Laser Doppler Flowmetry unit (Perimed, Stockholm, Sweden).

Blood Gas Analysis: Arterial blood gases were monitored to determine theeffects on pH, pCO2, and pO2. Blood was drawn from the arteriole cannulaand analyzed using a GEM Premier 3000 unit (Instrument Laboratory,Lexington, Mass.).

Study Design

The rats were randomly selected for various studies. Following surgerythe rats were allowed to stabilize for 30 minutes and a 15 minutesbaseline recording of all parameters (blood pressure, heart rate,cerebral blood flow, and renal blood flow) was obtained prior to theadministration of the pretreatment followed by IRL-1620 (Suc-[Glu 9, Ala11,15], American Peptide Co, Inc., Sunnyville, Calif.) or ET-1 (Ala1,3,11,15, RBI Sigma, Natick, Mass.). BQ-788, a selective endothelin Bantagonist, is the sodium salt ofN-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltriptophanyl-DNIe (see Proc. Natl. Acad. Sci. USA, 91, pp. 4892-4896(1994)) BMS is BMS 182, 874, which is a selective endothelin Aantagonist having a structure

Study 1

Group 1: Animals (n=4) were pretreated with saline 15 minutes (min)prior to administration of IRL-1620 (5 μg/kg, i.v.).

Group 2: Animals (n=4) were pretreated with BMS (5 mg/kg, i.v.) 15 minprior to administration of IRL-1620 (5 μg/kg, i.v.).

Group 3: Animals (n=4) were pretreated with BMS (15 mg/kg, i.v.) 15 minprior to administration of IRL-1620 (5 μg/kg, i.v.).

Group 4: Animals (n=4) were pretreated with BQ788 (1 μg/kg, i.v.) 15 minprior to administration of IRL-1620 (5 μg/kg, i.v.).

Study 2

Group 1: Animals (n=4) were pretreated with saline 15 min prior toadministration of ET-1 (0.75 μg/kg, i.v.).

Group 2: Animals (n=4) were pretreated with BMS (5 mg/kg, i.v.) 15 minprior to administration of ET-1 (0.75 μg/kg, i.v.).

Group 3: Animals (n=4) were pretreated with BMS (15 mg/kg, i.v.) 15 minprior to administration of ET-1 (0.75 μg/kg, i.v.).

Group 4: Animals (n=4) were pretreated with BQ788 (1 μg/kg, i.v.) 15 minprior to administration of ET-1 (0.75 μg/kg, i.v.).

Blood pressure, heart rate, cerebral blood perfusion, and renal bloodperfusion all were recorded for 2 hours following the final injection.Blood gases were analyzed prior to administration of any drugs, and at60 and 120 min following administration of IRL-1620 or ET-1. At the endof each experiment, the animals were euthanized with an overdose ofurethane i.v. In the figures, all data values are presented as mean±SEM.One-sample t-tests and one-way ANOVAs were used to test the differenceswithin and between the groups. A P value of P<0.05 was consideredsignificant.

FIG. 2. shows the effect of IRL-1620 (3 nmol/kg, iv) on cerebral bloodperfusion of urethane anesthetized rats using Laser Doppler Flowmetry.IRL-1620 significantly increased cerebral blood perfusion compared tobaseline.

FIG. 3 shows the effect of IRL-1620 (3 nmol/kg, iv) on cerebral andrenal blood perfusion of urethane anesthetized rat using Laser DopplerFlowmetry. IRL-1620 significantly increased cerebral blood perfusion anddecreased renal blood perfusion compared to baseline.

FIG. 4 shows the effect of IRL-1620 (3 nmol/kg, iv or 5 μg/kg, iv) onblood pressure and heart rate of urethane anesthetized rat. A transientdecrease in blood pressure and an increase in heart rate were observedwhich returned to normal quickly.

FIG. 5 shows the effect of IRL-1620 (5 μg/kg, i.v.) and ET-1 (0.75μg/kg, i.v.) on cerebral blood flow. IRL-1620 caused an increase incerebral blood flow of 12.79%, 18.17%, and 17.92% at 5, 10, and 15 min,respectively. This increase persisted for about 60 minutes. ET-1elicited no significant change in cerebral blood flow.

FIG. 6 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (ET_(A) antagonists) (1 μg/kg, i.v.) on the effect of IRL-1620(5 μg/kg, i.v.) on cerebral blood flow. BQ788 effectively blocked theeffect of IRL-1620 on cerebral blood flow, while the high dose of BMScaused a transient increase of 37.99% in cerebral blood flow 1 minuteafter administration of IRL-1620.

FIG. 7 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of ET-1 (0.75 μg/kg, i.v.) oncerebral blood flow. Pretreatment with the ET_(A) antagonists producedno significant change to the effect of ET-1 on cerebral blood flow.

FIG. 8 shows the effect of IRL-1620 (5 μg/kg, i.v.) and ET-1 (0.75μg/kg, i.v.) on renal blood flow. IRL-1620 elicited a decrease in renalblood flow of 16.94%, 15.05%, and 3.85% at 1, 5, and 10 minutes,respectively. This decrease persisted for approximately 15 minutes. ET-1elicited a decrease in renal blood flow of 40.27%, 50.10%, and 26.33% at1, 5, and 10 minutes, respectively. This decrease in renal blood flowalso persisted for about 15 minutes before returning to basal levels.

FIG. 9 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of IRL-1620 (5 μg/kg, i.v.) onrenal blood flow. BQ788 effectively blocked the effect of IRL-1620 onrenal blood flow, while the BMS caused a sustained decrease in renalblood flow up to 120 minutes after IRL-1620 administration.

FIG. 10 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of ET-1 (0.75 μg/kg, i.v.) onrenal blood flow. No pretreatment significantly altered the effect ofET-1 on renal blood flow.

FIG. 11 shows the effect of administration of IRL-1620 (5.0 μg/kg, i.v.)and ET-1 (0.75 μg/kg, i.v.) on mean arterial pressure. Mean arterialpressure decreased 33.32%, 23.88%, and 13.66% at 0.2, 0.5, and 1 minutefollowing administration of IRL-1620. Mean arterial pressure decreased43.16%, 37.80%, and 19.30% at 0.2, 0.5, and 1 minute, respectively,following ET-1 administration. The subsequent hypertension followingET-1 administration was recorded as an increase in mean arterialpressure of 12.72%, 25.56% and 28.49% at 5, 10, and 15 minutes,respectively

FIG. 12 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of IRL-1620 (5 μg/kg, i.v.) onmean arterial pressure. BQ788 effectively blocked the effect of IRL-1620on mean arterial pressure, while pretreatment with both doses of BMSproduced transient hypotension similar to that observed in animalspretreated with saline.

FIG. 13 shows the effect of pretreatment with BMS (5 and 15 mg/kg, i.v.)and BQ788 (1 μg/kg, i.v.) on the effect of ET-1 (0.75 μg/kg, i.v.) onmean arterial pressure. Pretreatment with BQ788 and BMS failed to alterthe effect of ET-1 on mean arterial pressure.

The following Table 1 shows the effect of IRL-1620 (5 μg/kg, i.v.) onheart rate. The values are expressed as mean ±SEM. Administration ofIRL-1620 when animals were pretreated with either saline or BMS (5 and15,mg/kg) caused an increase in heat rate. Pretreatment with BQ788effectively blocked the effect of IRL-1620 on heart rate. No significantalteration in heart rate was seen after administration of ET-1, with orwithout pretreatment.

TABLE 1 Time (min) Pretreatment 0 1 5 10 15 30 45 60 90 120 Saline 352 ±3 368 ± 6 351 ± 5  351 ± 4  353 ± 4  360 ± 3  370 ± 2  379 ± 3  392 ± 2 382 ± 8  1 μg/kg 344 ± 5 350 ± 6 345 ± 4  345 ± 4  347 ± 3  347 ± 4  350± 6  351 ± 9  351 ± 13 353 ± 13 BQ788 5 mg/kg 339 ± 6 365 ± 4 357 ± 13345 ± 14 345 ± 14 356 ± 12 367 ± 14 370 ± 14 350 ± 9  327 ± 12 BMS 15mg/kg  405 ± 11  425 ± 11 382 ± 13 380 ± 11 384 ± 11 387 ± 17 387 ± 21395 ± 19 393 ± 21 386 ± 18 BMS

The following Table 2 shows the effect of IRL-1620 and ET-1 on arterialblood gases. Neither IRL-1 620 nor ET-1 significantly affected bloodgases.

TABLE 2 pH pO2 pCO2 Time (min) 0 60 120 0 60 120 0 60 120 IRL-1620 7.32± 0.01 7.27 ± 0.01 7.29 ± 0.01 103 ± 3  99 ± 9 113 ± 6 42 ± 2 46 ± 6 32± 3 (5 μg/kg, i.v.) ET-1 7.32 ± 0.01 7.29 ± 0.01 7.27 ± 0.01 118 ± 4 110± 2 116 ± 5 34 ± 3 40 ± 4 38 ± 4 (0.75 μg/kg, i.v.)

The above tests and data show that administration of IRL-1620 produces asignificant increase in cerebral blood perfusion lasting about 60minutes. This effect can be blocked via pretreatment with ET_(A)receptor antagonist BQ-788.

1. A method of treating a stroke or cerebrovascular accident comprisingadministering a therapeutically effective amount of an ET_(B) receptoragonist to an individual in need thereof.
 2. The method of claim 1wherein the stroke or cerebrovascular accident is caused by thrombosis,embolism, or hemorrhage.
 3. The method of claim 1 wherein the ET_(B)receptor agonist is selected from the group consisting ofN-Succinyl-[Glu⁹, Ala^(11,15)] Endothelin 1, ET-3, sarafotoxin 6c,N-Ac-Ala(11,15)-endothelin-1 (6-21), Ala(1, 3, 11, 15)ET-1, and mixturesthereof.
 4. The method of claim 1 wherein the ET_(B) receptor agonistcomprises N-Succinyl-[Glu⁹, Ala^(11,15)] Endothelin
 1. 5. The method ofclaim 1 further comprising administering a therapeutically effectiveamount of a second therapeutic agent useful in the treatment of strokeor cerebrovascular accident.
 6. The method of claim 5 wherein the secondtherapeutic agent comprises a neuroprotective agent.
 7. The method ofclaim 6 wherein the neuroprotective agent is selected from the groupconsisting of a thrombolytic agent, an erythropoiesis-stimulating agent,an ET_(A) antagonist, an oxygen carrier, and mixtures thereof.
 8. Themethod of claim 7 wherein the thrombolytic agent comprises tissueplasminogen activator.
 9. The method of claim 7 wherein theerythropoiesis-stimulating agent is selected from the group consistingof erythropoietin, darbepoetin, epoetin alfa, and mixtures thereof. 10.The method of claim 7 wherein the oxygen carrier comprises ahemoglobin-based blood substitute, a perfluorocarbon-based bloodsubstitute, or a mixture thereof.
 11. The method of claim 7 wherein theET_(A) antagonist is selected from sulfosoxazole, clazosentan,atrasentan, tezosentan, bosentan, sitaxsentan, enrasentan, BMS 207940,BMS 193884, BMS 182874, J 104132, VML 588/Ro 61 1790, T-0115, TAK 044,BQ 788, TBC2576, TBC3214, PD180988, ABT 546, SB247083, RPR118031A,BQ123, and mixtures thereof.
 12. The method of claim 5 wherein theneuroprotective agent is selected from the group consisting ofargatroban, alfimeprase, tenecteplase, ancrod, sildenafil, insulin,insulin growth factor, magnesium sulfate, human serum albumin,caffeinol, microplasmin, a statin, eptifibatide, tinzaparin, enecadin,citicoline, edaravone, cilostazol, hypothermia, and mixtures thereof.13. The method of claim 5 wherein the ET_(B) agonist and the secondtherapeutic agent are administered simultaneously.
 14. The method ofclaim 13 wherein the ET_(B) agonist and the second therapeutic agent areadministered from a single composition.
 15. (canceled)
 16. The method ofclaim 5 wherein the ET_(B) agonist and the second therapeutic agent areadministered separately.
 17. The method of claim 16 wherein the ET_(B)agonist is administered prior to the second therapeutic agent. 18.(canceled)
 19. The method of claim 1 wherein the ET_(B) agonist isadministered in an amount of about 0.005 to about 500 micrograms perdose.
 20. The method of claim 1 wherein the ET_(B) agonist isadministered in an amount of about 0.005 to about 50 micrograms perkilogram per min infusion.
 21. A kit comprising: (a) a packagedcomposition comprising an ET_(B) agonist; (b) optionally, a packagedcomposition comprising a second therapeutic agent useful in a treatmentof stroke or other cerebrovascular accident, (c) an insert providinginstructions for a simultaneous or sequential administration of (a), or(a) and (b), to treat the stroke or other cerebrovascular accident in ahuman; and (c) a container for (a), (b), and (c).
 22. (canceled)
 23. Acomposition comprising (a) an ET_(B) agonist, (b) a neuroprotectiveagent, and (c) an optional excipient and/or pharmaceutically acceptablecarrier.